PDE3 and the Regulation of Myocardial Contractility
Cyclic nucleotide phosphodiesterases regulate intracellular signaling by hydrolyzing cAMP and/or cGMP. By blocking their hydrolysis, phosphodiesterase inhibitors potentiate cyclic nucleotide-mediated signaling. Eleven families of these enzymes have been described1. Two subfamilies, PDE3A and PDE3B, have been identified2,3. Myocardial contractility is regulated by PDE3A, and Pde3a ablation in mice increases the phosphorylation of two sarcoplasmic reticulum proteins involved in intracellular Ca2+ cycling: phosphorylation of phospholamban (PL or PLB) which stimulates the activity of the Ca2+-transporting ATPase of the sarcoplasmic reticulum (SERCA2), increasing Ca2+ uptake during diastole, and phosphorylation of ryanodine-sensitive Ca2+ channels which increases Ca2+ release from the sarcoplasmic reticulum during systole4-6 (FIG. 1). These changes in protein phosphorylation augment myocardial contractility by increasing the amplitude of intracellular Ca2+ transients6. The role of PDE3A in regulating Ca2+ uptake by the sarcoplasmic reticulum is likely to be linked to its integration into an intracellular signaling complex that includes SERCA2, AKAP18, phospholamban and PKA6.
PDE3 Inhibition in Heart Failure
In dilated cardiomyopathy, decreases in myocardial β-adrenergic receptor density, together with increases in Gαi and β-adrenergic receptor kinase activity, attenuate the stimulation of adenylyl cyclase by catecholamines, leading to decreases in myocardial cAMP content and intracellular Ca2+ transient amplitude7-16. PDE3 inhibitors are used to ‘overcome’ this reduction in intracellular cAMP content and increase cAMP-mediated signaling in failing myocardium. In the short term, PDE3 inhibitors raise cardiac output and lower left ventricular filling pressures17-23. With long-term administration, however, these benefits are outweighed by an increase in sudden cardiac death24. While our knowledge regarding the mechanisms is limited, they appear to be separate from those that augment contractility: an increase in SERCA2 activity, the consequence of phospholamban phosphorylation (FIG. 1), has anti-arrhythmic effects in animal models of ischemia/reperfusion and chronic heart failure25,26. Pro-apoptotic consequences of PDE3 inhibition, though, are likely to contribute to pathologic remodeling in dilated cardiomyopathy27. PDE3 inhibition in rats and Pde3a ablation in mice lead to increases in the phosphorylation of cAMP response element-binding protein (CREB) and consequent increases in the expression of inducible cAMP early repressors (ICER's), promoting apoptosis (FIG. 1)6,28,29. Conversely, PDE3A overexpression in mice reduces ICER, increases Bcl-2 expression and reduces apoptosis following ischemia/reperfusion injury (but reduces myocardial contractility)30.
Alternative Path Ways for cAMP-Mediated Protein Phosphorylation
Until now, studies of the contractile and pro-apoptotic effects of PDE3 inhibition have focused principally on substrates of PKA, which is activated directly by cAMP. More recently, it has become clear that the effects of cAMP are also mediated by guanine-nucleotide-exchange proteins activated by cAMP (Epacs), which influence protein phosphorylation through diverse mechanisms (FIG. 2)31,32. In cardiac myocytes, Epac activation augments contractility through signaling pathways that increase the phosphorylation of ryanodine-sensitive Ca2+ channels of the sarcoplasmic reticulum by Ca2+/calmodulin-activated protein kinase II (CamKII)—which was seen in pde3a−/− mice1—and of sarcomeric proteins such as cardiac myosin-binding protein C and troponin I by CamKII and protein kinase C (PKC)32-36. Epac activation also has pro-hypertrophic actions that result, at least in part, from the activation of CamKII, as well as the protein phosphatase calcineurin37,38. These observations indicate that changes in intracellular cAMP are likely to affect the phosphorylation of a large number of proteins that may contribute to the beneficial and adverse effects of PDE3 inhibition. A recent study of responses to β-adrenergic receptor activation in mouse embryonic fibroblast cells showed both increases and decreases in protein phosphorylation39, and, in our experiments, exposure of cultured cells to agents that stimulate cAMP-mediated signaling resulted in both increases and decreases in protein phosphorylation, covering both PKA and non-PKA sites. Interactions between PDE3B and Epac have been identified in vascular smooth muscle myocytes40, and peptides that disrupt this interaction activate Epac and lead to the activation of phosphoinositide-3-kinase-γ (PI3Kγ), extracellular signal-related kinase (ERK) and protein kinase B (PKB, also known as Akt)41, but the role of PDE3 in regulating Epac-mediated protein phosphorylation in cardiac myocytes remains unexplored. cAMP can also regulate L-type Ca2+ channels directly42, and Epac activation induces other responses in addition to protein phosphorylation31.
Individual Phosphodiesterases Regulate cAMP-Mediated Signaling in Distinct Intracellular Compartments of Cardiac Myocytes
In this context, it is noteworthy that cAMP content is regulated differentially in spatially and functionally distinct compartments of cardiac myocytes, a phenomenon referred to as the ‘compartmentation’ of cAMP-mediated signaling. It has long been known that exposure to β-adrenergic receptor agonists increases cAMP content in cytosolic and microsomal fractions of cardiac muscle and augments contractility, while exposure to prostaglandin E1 (PGE1) increases cAMP content only in cytosolic fractions, without inotropic effects43,44. This compartmentation of cAMP-mediated signaling is altered in a rat model of heart failure following a redistribution of β-adrenergic receptor subtypes within cell membranes45, and is a feature of the pathophysiology of dilated cardiomyopathy in humans (both ischemic and nonischemic), where the reduction in cAMP content is much more pronounced in microsomes than in cytosolic fractions (FIG. 3)15.
Over the past decade, the prominent involvement of phosphodiesterases in this compartmentation has become apparent. In rat cardiac myocytes, β-adrenergic receptor agonists induce increases in intracellular cAMP content that are highly localized46. Individual phosphodiesterases, which are targeted by protein-protein interactions to specific intracellular domains, have distinct roles. In rat heart, PDE4 has a greater role than PDE3 in regulating glucagon and catecholamine-mediated increases in intracellular cAMP content, while PDE3 has a greater role in regulating forskolin-induced increases47,48. PDE2 has a major role in regulating β-adrenergic receptor-mediated increases in intracellular cAMP content but only a small role in regulating forskolin-induced increases, and PDE2 and PDE3 regulate ‘opposing’ effects of cGMP on cAMP-mediated signaling in rat heart in functionally separate compartments49,50.
These studies focused on phosphodiesterase families, but individual isoforms within a family have precise roles in specific intracellular microdomains. This has been examined extensively in the PDE4 family. PDE4D3 is present in multiprotein complexes regulating KCNQ1/KCNE1 K+ channels and ryanodine-sensitive Ca2+ channels51,52. The latter are hyperphosphorylated in Pde4d−/− mice, leading to abnormalities of sarcoplasmic-reticulum Ca2+ release associated with arrhythmias and the development of dilated cardiomyopathy52. In contrast, experiments in Pde4d−/− and Pde4b−/− mice showed that the stimulation of L-type Ca2+ currents by β-adrenergic receptor agonists is controlled specifically by PDE4B53. These unique roles for PDE4 variants derive principally from the differences in their intracellular localization, which in turn reflect the distinct protein-protein interactions through which they are recruited to intracellular signaling complexes with a range of proteins, including AKAP's, β-arrestins, Src, Lyn and Fyn54,55.
Multiple Isoforms of PDE3 are Expressed in Human Myocardium
In human cardiac myocytes, the PDE3A gene gives rise, through a combination of transcription and translation from alternative sites, to several isoforms whose amino-acid sequences are identical save for the presence of different lengths of N-terminal sequence that are involved in intracellular localization, protein-protein interactions and allosteric regulation of catalytic activity, which resides in the C-terminus (FIG. 4)56. PDE3A1, a 136-kDa protein, has a unique N-terminal extension containing hydrophobic loops that insert into intracellular membranes57,58, and three known sites of phosphorylation, S293, S312 and S42859-61. PDE3A2, a 118-kDa protein transcribed from a downstream site in exon 1, lacks the N-terminal extension and S293. PDE3A3, which is translated from a downstream site in the PDE3A2 mRNA, is a 94-kDa protein that lacks all of these phosphorylation sites. The PDE3B gene gives rise to a single 146-kDa protein, whose domain organization resembles that of PDE3A1. Its C-terminal catalytic region is highly homologous to that of PDE3A1, and it contains an N-terminal hydrophobic sequence and phosphorylation sites similar to two of the three sites identified in PDE3A162,63. In view of their C-terminal sequence identity (for PDE3A1, PDE3A2 and PDE3A3) and homology (PDE3B), all four isoforms are similar with respect to their basal catalytic activity and sensitivity to existing PDE3 inhibitors64.
The Potential Opportunity and its Clinical Impact
The American Heart Association estimates that 5.7 million Americans have heart failure. Each year, >550,000 new cases are diagnosed, among which ˜50% involve impaired contractility. The annual hospitalization rate is >1 million, and annual mortality is >270,00065,66. An agent that could inhibit cardiac diseases such as hypertrophy and increase survival would represent a major advance, and its clinical impact would be immense. While the benefit might be greatest for patients with advanced heart failure who are poor candidates for ventricular assist devices, artificial hearts and heart transplantation—which includes the ever-increasing population of aging patients with comorbidities that are contraindications for surgery—a large proportion of patients with NYHA class 3 or 4 symptoms could be expected to benefit. Our discovery provides a novel approach to this enormously important clinical problem.